Thioredoxin (Trx) was first recognized in the early 1960s as the reductant used by a variety of enzymes. Two Trxs have been identified in various species including humans, one cytosolic (Trx1) and one mitochondrial (Trx2). Trx was first identified as a hydrogen donor for enzymes involved in reductive reactions. However, the Trxs also play an important role in maintaining the reduced environment in the cells through thiol- disulfide exchange reactions, which is ideally suited to control protein function via the redox state of structural or catalytic SH groups. Therefore, the Trxs could play an anti-aging action either through their ability to reduce the leve of oxidative stress/damage or through alterations in redox-sensitive signaling, which has diverse effects on pathophysiology. Over the past three years, we have conducted the first detailed study on the effect of overexpressing or down-regulating Trx1 or Trx2 on aging. Interestingly, we found that the Trx2Tg mice that overexpress Trx2 in all tissues during aging showed a significant extension of lifespan (25.6%) compared to wild-type mice, although we did not observe a significant increase in survival of the Trx1Tg mice, when the Trx1 overexpression was maintained over the lifespan. The extension of lifespan of Trx2Tg mice was correlated to less reactive oxygen species (ROS) production from mitochondria and less oxidative stress. These data indicate that overexpressing Trx in the mitochondria may be more important than in the cytosol on aging because mitochondria are a major source of ROS. When we tested the effects of reduced levels of thioredoxin in cytosol or mitochondria on aging, we surprisingly observed the reversed effects, i.e., a significant increase in survival of the Trx1KO mice (12.6%) compared to wild-type mice while the Trx2KO mice showed little effects on lifespan. The extension of lifespan of Trx1KO mice was associated with fewer cancers compared to wild-type mice at 22-24 months of age. These data indicate that reduced cancer in the Trx1KO mice could be one of the contributing factors of extended lifespan. These interesting observations led us to these two questions: 1) Do the Trx2Tg x Trx1KO mice, which increase Trx2 in mitochondria and reduce Trx1 in cytosol, show additive anti- aging effects; and 2) Do overexpression of Trx2 and/or downregulation of Trx1 attenuate age-related pathology? The goal of this application is to address these two questions by using transgenic mice that overexpress Trx2, heterozygous knockout (KO) mice that down-regulate Trx1, and their offspring that increase Trx2 in mitochondria and reduce Trx1 in cytosol (Trx2Tg x Trx1KO mice). Using these mice, we will test the following hypothesis: overexpression of thioredoxin in mitochondria (Trx2) along with down-regulation of thioredoxin in cytosol (Trx1) will show a greater extension of lifespan and a greater reduction in age- related pathology compared to the overexpressing Trx2 or down-regulating Trx1 alone, and the extension of lifespan will occur because of additive effects of independent mechanisms, i.e., protection against oxidative stress/damage in mitochondria and attenuation of pathology, e.g., cancer. Specific Aim 1: To measure the effect of overexpressing Trx2 and/or down-regulating Trx1 on aging and age- related physiological changes in mice. Specific Aim 2: To measure the effect of overexpressing Trx2 and/or down-regulating Trx1 on age-related pathology in mice. Specific Aim 3: To measure the effect of overexpressing Trx2 and/or down-regulating Trx1 on the antioxidant status, the age-related accumulation of oxidative damage, the mitochondria function and the reactive oxygen species production, and the redox status in various tissues of mice. Specific Aim 4: To measure the effect of overexpressing Trx2 and/or down-regulating Trx1 on redox-sensitive signaling in various tissues of mice.